Integration of foreign DNA into the genome of organisms and cell lines is a widely utilized method for interrogation and manipulation of biological systems. Traditionally, transgene insertion is targeted to a specific locus by provision of a plasmid carrying a transgene, and containing substantial DNA sequence identity flanking the desired site of integration. Spontaneous breakage of the chromosome followed by repair using the homologous region of the plasmid DNA as a template results in the transfer of the intervening transgene into the genome. See, e.g., Koller et al. (1989) Proc. Nat'l. Acad. Sci. USA 86(22):8927-8931; Thomas et al. (1986) Cell 44(3):419-428. The frequency of this type of homology-directed targeted integration can be increased by up to a factor of 105 by deliberate creation of a double-strand break in the vicinity of the target region (Hockemeyer et al. (2009) Nature Biotech. 27(9):851-857; Lombardo et al. (2007) Nature Biotech. 25(11):1298-1306; Moehle et al. (2007) Proc. Nat'l Acad. Sci. USA 104(9):3055-3060; Rouet et al. (1994) Proc. Nat'l Acad. Sci. USA 91(13):6064-6068.
A double-strand break (DSB) or nick for can be created by a site-specific nuclease such as a zinc-finger nuclease (ZFN) or TAL effector domain nuclease (TALEN), or using the CRISPR/Cas9 system with an engineered crRNA/tract RNA (single guide RNA) to guide specific cleavage. See, for example, Burgess (2013) Nature Reviews Genetics 14:80-81, Urnov et al. (2010) Nature 435(7042):646-51; United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20090263900; 20090117617; 20100047805; 20110207221; 20110301073 and International Publication WO 2007/014275, the disclosures of which are incorporated by reference in their entireties for all purposes. In many organisms, transgene insertion can be accomplished via homology-directed repair (HDR) processes, which require the inserted transgene to include regions of homology to the site of insertion (cleavage). However, some organisms and cell lines lack traditional HDR process and targeted integration occurs primarily via the homology-independent non-homologous end joining (NHEJ) DNA repair machinery. As such, to date, in organisms and cell lines (e.g., CHO cells) that are recalcitrant to HDR processes, only relatively short (<100 bp) oligonucleotides have been integrated via homology-independent pathways following nuclease-mediated cleavage of the target locus. See, e.g., Orlando et al. (2010) Nucleic Acids Res. 38(15):e152 and U.S. Patent Publication No. 20110207221.
Thus, there remains a need for compositions and methods for homology-independent targeted integration of transgenes, including larger transgenes, directly into the site of cleavage, for example in organisms and cell lines that lack, or are deficient in, traditional homology-driven approaches.